Process of producing butyl alcohol



Patented May 9, 1933 UNITED STATES PATENT w ALEXANDER IZSAK, or CENTRAL PARK,'1\TEW JEBSE'Y, AND: Eo s'n 'J... EuNx, E WILMINGTON, DELAWARE, AssreNoRsLY MESNE AssrGNmm-rs, Iron 1'. nuiroN'r DE NEMOURS AND COMPANY, or wrLmrNeroN, D LAwAE AconroRArroNor DELAWARE No Drawing. Continuation of application Serial PROCESS or rEonUcrNG 'IBUTYL ALco'noL- ma as, 1931. Serial No. 540,628."

This invention relates to a processof producing normal butyl alcohol, acetone, and

isopropyl alcohol by the fermentation of non-amylaceous carbohydrates, and more particularly to the production of such prodproduce satisfactory yields "of these solvents by the fermentation of canemolasses without success. It has become rather common practice to produce such solvents by fermentation of cereal mashes with theorganism variously known as Bacillus granalobact'er pectinooov factory results may be obtained in the absence of amylaceous vegetable materlal, In

mmBeyerinck and Van Delden (British Patent 4845 of 1915, to C. Weizmann) 0Z,ostm'd- {um acetobutyljwm (British Patent 315,002 to Jan Augusto Viljoen), and similarlyjbyother names. This organism is primarily a starch fermenter, and while it will, under certain laboratory conditions, ferment various sugars, as wellas starch, with apparently satisfactory results, attempts to ferment sugars directly on a commercial manufactur-' ing scale using this particular organism, have not beensuccessful, principally because of the peculiar physiological properties possessed by theorganism. I

With a view of overcoming the difficulties previously encountered, Waters (U. S."Patent 1,546,694) proposes the addition $0111 0- lassesmashes of vegetable protein materlal,

' such as corn gluten {Robinson (U. S. Patent 1,510,526) proposes a pretreatment of the molasses with activated charcoal; Viljoen (British 315,002 supra) uses a very large inoculum, and treatsthe mash so as to secure predigestion of the proteins present or added; Pike and Smyth (U. S. Patent 1,655,435)

have designated the organism Olostridz'um but z m'cum Prazmowski, and assert that satisso far as we are, aware, none of these processes have been. entirely commercially successful primarily because the organ1sm,. notwithstanding attempts at acclimatization to a.

sugar medium-instead of a starch medium, remains essentially a starch fermenter, and cannot be depended upon to produce its characteristic reaction in the presence of sugars, or n a sugar medium.

- In further pursuance of the general investigation which led" to the isolation of B. .saco'hambutg ZMM beZa '(Izs'ak U: 'S. Patent No. 1,725,083) we have now isolated a second organismbelonging to the same group, which we have named 'OZos MJi m sqccfl arobutylic'um-gamma. As described in the patent above referred to,"the object of the former invention wa's'th'e productionfof 'butyl and isopropyl alcohols in'the' main, along with small quantities of acetone. The new organism which wehave now isolated, when used to ferment, for example cane molassesfblack times as much acetone as isopropyl.

Aside from the variation in thei relative No. 4sa,914, filed July 22, 1939. 'Ihlsapplicat ion inq proportions of the'en-d prodn'cts' of the fermentation, and the methods of their isolation, these two organisms are practically indistin guishable from each other. {Neither can forment cereal starch to any practical extent; 1

both can producejhigh yields of'solvents in the presence Of G X CGSS calcium carbonate.

Neither culture can be used in the commercially operating processes for the production of butyl alcohol and'acetone directly from corn meal or other cereal products as now practiced. One difl'erence which is of importance from an industrial standpoint is that with ordinary black-strap molass the usejof calcium carbonate or other simil r neutralizing agents is desirable, when beta is used, while under the same conditions the addition of calcium carbonate to gamma fermentations' seems to be without either favorable or adverse efiect.

According to our present inventionwe have now discovered a new'process for producing mainly butyl alcohol "and acetone with other products by means of an organism not here-- tofore isolated, or used for this purpose. This new organism is capable of fermenting the sugars in a solution of black-strap mo- 5 lasses, or in other non-amylaceous mashes with satisfactory ields of butyl alcohol and acetone, without t e addition of foreign protein or vegetable material, and without pretreatment of the solution to be. fermented other than sterilization which is essential in any case. This, organism, to which we .have

given the name Glostrz'dium sdccharobutylicum-gamma is characterized jiniaccordance with" the descriptive .chart of the Society of I American Bacteriologists, as follows:

sacckaro butg lz 'cumearly short chain formation.

Size: 6-20 microns X 2-3microns.

End's: Rounded Stain: Will not stain with methylene blue or gentian violet readily Gram stain variable ,2. Sporangia:

Media used-6 Brix solution of blackstrap molasses, partly neutralized with calcium carbonate; pH 5.6; temp. 34 (3.; age 60 hours. Form: Oval- Spores: Sub-terminal to terminal Limits of Size: 2 to 4 microns. Spores not stain readily with methylene blue.

II. Cultural Eeatures.

1. Nutrient agar slant: no growth (on aerobic surface). a

Nutrient agar in deep stab: no growth along stab until depth of 1' cm. is reached. Below 'this point growth on puncture uniform, papillate. III. Physical and biochemical features. 50 Substances fermented with gas evolution 12 116m 40 hours xx Strong Form: Short and long rods, occasionalxxx Very strong xxxx Strongest Enzyme production:

Amylase, very weak Organism dies on repeated transfers in corn meal suspensions. Products of fermentation Liquid: n- Butyl ralcohol (-80%).

Actone (18-34%) Isopropyl alcohol -('1-2%) Traces. of ethylalcoh and other compounds.) Ij-ji H Gaseous: CO Y It] will be seen by comparing tlredescripl tion'of our new species oforganisniiwith that of the. organism used commercially, for the production of butyl'alc'ohol; and acetone from.

v cereal mashes as given in the 'literatm'e pr'eviously cited, that it differs specifically from the latter in its inability toliquefy and/or ferment the major portion'of a cereal starch mash. Its gross characteristics,"morphology, and its fermentation reactionsexcept toward cereal starch, as will be noted, are in general, similar to those of the latter organism; it .is, however, a distinct species, and cannot be substituted for the latter organism in the grain mashes in whichthe latter organism-is commercially employed. 4

This-new organism which we prefer to call Olostrz'dium saccharobuty lioum-gamma is also specifically different from the foregoing commercial organism, known as Olostfidz'um aaetobutylz'cum, Glostfldium butyricum, etc., in other-important characteristics. For example, in a sugarsolution containing alkaline material, such as calcium carbonate,or in such solutions where the acidity iscontinuously reduced by the addition of soluble alkalies, the OZostridiwn will produce large amounts of butyric acid, so much so in fact as to almost eliminate theforma- Zymologie pure et applique,vol. 1, No. 3 ;Ann.

Brass. et Dist.,'1927. vol. 25, pp. 321-327, 343-347, 359-362). Weizmann and Spiers (British Patent 164,762\ have disclosed the production of butyric acid by this organism in media containing calcium carbonate or other alkalies.

In contrast with this behavior, we have found that our new organism, Ulost'rz'dium saacharob'utyZ'Zcwn-gamma, will ferment sag ar solutions in the presence of calcium carbonate, without serious or-even detectable reduction in the yield of solvents, and without the production of more than traces of hutyric acid. On the other hand, the presence of calcium carbonate or other similar material sometimes used in the mash to be fermented is not only not essential, but may be considered useless and without effect, al-' .though not harmful. i

The fact that our organismis new and differs markedly from other organisms capable of producing butyl alcohol that have been hitherto describedor employed'in the artwill be more readily understood from the follow-' ing description of the method employed and the principles involved, in, isolating and developing it explained in greater detail hereinafter. A I It is a well known principle that starch is a. carbohydrate-stored up by many higherv plants in a form insoluble in water for the future use of themselves or their hydrate, generically known as sugar. There are in nature other organisms incapable themselves of, or rarely producing starch which are, however, capable of producing the hydrolytic agent (amylase) which changes insoluble starch into soluble sugar. They are thus enabled to appropriate or their own uses a food stored up by another organism. Such a group of organisms is exemplified by those butyl alcohol producing organisms which are described in the literature and used in the industry. There is still another class of lower organisms which, while requiring water-soluble carbohydrates, dproduced by other living organisms as a foo are not able to produce the amylase which would permit them to appropriate insoluble starch which may be in their immediate environment. Examples of organisms in this general class are the well known yeasts used in industry for the productionof ethyl alcohol. Such organisms often feed at the same table with other low forms upon food which neither of the mess mates alone is capable of itself roducing. Under such circumstances one o the organisms of the lower forms may be able to produce such a saccharifying enzyme (amylase) as will yield from available starch as much sugar asiboth need. It will thus be apparent that many low forms of organisms are capable of growing on carbohydrates which are produced either originally in the water soluble form or are brought into such form by the activities of another organism.

The problem that-confronted us was to ascertain the existence, by isolation, of any organisms capable of producing butyl alcohol in commercial quantitieswhich might belong to the second unexplored class of nonamyla'se producing forms. One of the early results of this study was the isolation of such an organism which was found capable of A producing butyl alcohol, isopropyl alcohol,

and acetone from soluble carbohydrates.

Such .a culture is the essential organism described and claimed in'Izsak U. S. Patent,

We have now discovered this secondfnew organism (Ulostm'dium saccharo butylicum 3 gamma) similarly incapable offermenting cereal starch unaided. This organism,--like many heretofore described, produces'chiefly butyl alcohol and acetone, but differsfrom them so markedly that it cannot "be used in commercially existing processes without fundamental changes in equipmentand pro;

cedure as hereinbefore described.

In the isolation of this organism from its original habitat, rice, such methods of heat treatment, culture in media of varying pH concentrations, carbohydrate source, 'etc.,

were employed as would tend to favorv the.

development of organisms of this particular type if they existed. i

The following illustrative example-of one method of isolating of this organism is as follows: i

Rice grains (a source of starch) distribut-.

ed in several containers, were heatedin boil ing water for minutes; this naturally eliminated nearly all of the living organisms on the starch since most ,micro-organisms are incapable of with standing such treatment. Next, sufficient concentrated lactic acid was added to the -medium to bring the pH to approximately 3.0. This step provided a still further limiting factor which would further restrict the number and char? acter of the surviving organisms which'could later develop. A'second function of the add ed acid was to transform the insoluble starch to a soluble form.- To-facilitate this-transformation the rice suspension was incubated at 63 C. for 48 hours,'this producing hydrolytic products grading through soluble starch to sugars. Calcium carbonate-was then added in excess," the suspension again heated to 100 (1., and returned to the 63 C. incubator for another 24 hours. Finally the containers prepared as above were incubated at 32 C. to permit possible -fermentation to set in, and a large percentage of them developed fermentation, the active organ; ism being Olostrz'dz'mn' saclumiob'mh Zz'cumgamma. 5-

No plating is required to purifytheculture on account of the rigorous selective proclgss used in the isolation, but heating to C. or 15 minutes and reincubating several times is all that is necessary. This last step is applied to spore laden-cultures, after several the process of producing butyl "alcohol, etc, by an organism isolated and purified in the.

manner described above, as other methods will suggest themselves from the foregoing. As a further illustration of our invention the following example is given of the methodof using this organism, but it is to be un p.15. 24.hours.'. .At the end of this time, this laboratory culture was added under asepticconditions, to 150 gallons of a similarly ster ilized solution of black-strap molasses in a suitable small fermenter, or seed tank. This seed culture was in turn kept at 33 to-38 C.

.for about 24 hours, whereupon it was used to inoculate a similar mash having a volume of approximately 4,850 gallons. This mash was then allowed to ferment at the above specified'temperature for about 72 to. 120 hours, whenfermentation had ceased. The fermented beerwas then found to contain about 1 to 1.25 per cent of solvents (i. e., norfrom the beer by well known methods,

mal butyl alcohol and acetone, and isopropyl alcohol, plus possible traces of other compounds) by volume, which may be distilled There was obtained approximately 50 to 65 gallons of anhydrous solvents, representing about 32 per cent by weight of the original carbohydrates (calculated as invert sugar) in the original mash. The amounts ofv solvents obtainable in this way will, of course,

vary, depending upon the original sugar con.-;

mash, a culture of a micro-organism capable of producing, unaided, from the carbohy- I tent. Substantially" greateryields than 32 per cent may be obtained when careful attention is given to the technique of .the

process.

,While-the preferred concentration of the mash is from 30 to 40 grams of sugar per litre, any concentration up to about 75 grams per litre may be used. The preferredtemperature range is between 33 and 38 0., but temperatures of from 25 to 40 C., or even higher are possible. .Outside of the preferred range of temperatures, however, the fermentation becomes too. slow to be practical economically.

The molasses solution constituting the mash to be fermented need not necessarily be clarified, or treated with activated carbon, nor is it necessary that the hydrogen ion concentration be adjusted to any other thanthat resulting from the acids contained in the molasses itself, which is usually between a pH of 5 toa pH of 6. The addition of foreign proteins, such for example as-corn gluten, soy bean meal, etc., carbon ammonium salts, and phosphates is unnecessary as is also the addition of calcium carbonate or an other alkaline material, although these su stances may be added if desired.

While the foregoing description and examples illustrates certain embodiments of our invention, particularly as applied to the fermentation of black-strap molasses, we do not intend to be limited thereto, as sugars from other sources may equally well be used, such for example as commercial dextrose or maltose, cane sugar, whey, and beet molasses, the appropriateand necessary nitrogeneous material and salts in the case ofpure sugars' being supplied to replace the nutrients normally present in molasses. It will, therefore, be apparent that We do not intend to limit our invention except as indicated in the appended claims.

We claim: y 1. The process which comprises adding to a sterilized, non-amylaceous, water-soluble carbohydrate mash, aculture of bacteria which are incapable, unaided,-of fermenting cereal starch, which are sufficiently heat resistant in the spore form to withstand a temperature of 100 C. for approximately five minutes and .are capable of producing butyl alcohol and acetone by the fermentation of the carbohydrates in such a mash, and maintaining the thus seeded mash at a temperature sufficient to bring about active fermentation.

2. The process of claim 1 in which the source of the carbohydrate is black-strap molasses. r

3. The process of claim 1 in which the mash is maintained at a temperature of 24 C. to 40 C.

4. The process that comprises adding to a water-soluble, non-amylaceous carbohydrate drate in the mash,butyl alcohol and acetone in a ratio substantially greater than two parts of butyl alcohol to one part of acetone.

5. The process which comprises adding a "culture of a herein described micro-organism to a sterilized mash of non-amylaceous,water-soluble carbohydrate and maintaining the mash at a temperature suflicient to bring about active fermentation. V 6. The process of claim 5 in which the source-of the carbohydrate is ablack-strap molasses mash.

V 7. The process of claim 5 in which the temperature of .the mash is maintained between 24 C. and 40 C. during fermentation.

8. The process which'comprises adding a culture of a hereinbefore described microorganism C'Zostrz'dium sacokarobutg lz'mmigamma to a sterilized mash containing approximately 0.5 to 7 .5 per cent. of nonamylaceous, water-soluble carbohydrate, and Y maintaining the seeded mash at a temperature suflicient to bring about active fermentation. I v

9. The process of claim 8 in which the sugar' concentration of the mash is between 3 and 6 per cent.

10. The process of claim 8 in which the source of the carbohydrate is black-strap mov lasses.

11. The process of claim 8 in which the fermentation is carried out in the absence of foreign protein and vegetable matter.

12. The process which comprises adding a 10 culture of the hereinbefore described microorganism C'Zostridium 'sacckarobutylicumgamma to a sterilized medium containing non-amylaceous, water-soluble carbohydrate, maintaining the seeded mash at a temperature sufficient to bring about active fermentation, and recovering from the fermented mash butyl alcohol, acetone, and isopropyl alcohol, which have been formed therein 1n decreasing ratios respectively.

13. The process of claim 12 in which the source of the carbohydrate is black-strap molasses.

14. The process of claim 12 in which the sugar concentration of the carbohydrate is between 0.5 and 7.5 per cent.

15. The process of claim 12 in which the sugar concentration of the carbohydrate is between 3 and 6 per cent.

16. The process of claim 12 in which the temperature of the mash is maintained at 24 C. to 40 C.

17. The process of claim 12 in which the fermentation is carried out in the absence of foreign protein and vegetable matter.

hol and acetone by fermentation, of preparing suitable cultures which comprise heating a plurality of samples of rice grains in boiling water for approximately thirty minutes, adding suflicient concentrated lactic acid to the medium to bring the pH to approximatel 3.0, incubating the suspension at about 63 5., adding calcium carbonate to the suspension, heating the suspension to approximately 100 0., and again further incubati the samples.

19. T 0 process of producing butyl alcohol, acetone and isopropyl alcohol which comprises causing the fermentation of a nonamylaceous carbohydrate mash by means of the organism Glostridz'um sacckarobutylicmm gamma.

. 20. The process of producing butyl alcohol and acetone which com rises causing the fermentation of a sugar so ution by means of the organism Ulostridimn 'sacckarobutglicamgamma.

j 21. The process of producing butyl alcohol and acetone which comprises causing the fer- 30 mentation of a molasses mash by means of the organism OZostm'dz'wn sacckarobutglz'awmgamma.

22. The process of claim 21 in which the fermenting solution is maintained at a tem- B perature of from 33 to 38 C.

18. In the process of producing butyl alco the steps Y 23. The process of producing butyl alcohol, acetone and isopro yl alcohol w ich comprises causing the ermentation of a sugar solution of the t pe found in molasses mash, malt or whey by means of the organism Glostfidiwn saccharobutgZz'cam-gamma.

24. The process of claim 21 in which the mash has a sugar concentration of 3 to 5%.

25. The process of producing butyl alcohol and acetone which comprises causingthe fermentation of a molasses mash free of foreign protein and vegetable matter by means of the organism Olostrz'dium sacaharobutglz'awmgamma.

26. The process of producing butyl alcohol and acetone which comprises causing the fermentation of a sugar solution containing foreign protein material by means of the organism. Clostfidiu/m saccharobutgliml/mgamma.

27. The process of producing butyl alcohol, acetone and isopro yl alcohol which comprises causing the ermentation of a blackstrap molasses free of foreign protein and vegetable matter by means of the organism Oloszffidz'mn saccharobatglicum-gam/ma.

In testimony whereof we aflix our signatures.

ALEXANDER IZSAK. FOREST. J. FUNK. 

